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Single-cell genomics technology has transformed our understanding of complex cellular systems. However, excessive cost and a lack of strategies for the purification of newly identified cell types impede their functional characterization and large-scale profiling. Here, we have generated high-content single-cell proteo-genomic reference maps of human blood and bone marrow that quantitatively link the expression of up to 197 surface markers to cellular identities and biological processes across all main hematopoietic cell types in healthy aging and leukemia. These reference maps enable the automatic design of cost-effective high-throughput cytometry schemes that outperform state-of-the-art approaches, accurately reflect complex topologies of cellular systems and permit the purification of precisely defined cell states. The systematic integration of cytometry and proteo-genomic data enables the functional capacities of precisely mapped cell states to be measured at the single-cell level. Our study serves as an accessible resource and paves the way for a data-driven era in cytometry.

Preclinical research of myelodysplastic syndromes (MDSs) is hampered by a lack of feasible disease models. Previously, we have established a robust patient-derived xenograft (PDX) model for MDS. Here we demonstrate for the first time that this model is applicable as a preclinical platform to address pending clinical questions by interrogating the efficacy and safety of the thrombopoietin receptor agonist eltrombopag. Our preclinical study included n= 49 xenografts generated from n= 9 MDS patient samples. Substance efficacy was evidenced by FACS-based human platelet quantification and clonal bone marrow evolution was reconstructed by serial whole-exome sequencing of the PDX samples. In contrast to clinical trials in humans, this experimental setup allowed vehicle- and replicate-controlled analyses on a patient–individual level deciphering substance-specific effects from natural disease progression. We found that eltrombopag effectively stimulated thrombopoiesis in MDS PDX without adversely affecting the patients’ clonal composition. In conclusion, our MDS PDX model is a useful tool for testing new therapeutic concepts in MDS preceding clinical trials.

Blood formation is believed to occur through stepwise progression of haematopoietic stem cells (HSCs) following a tree-like hierarchy of oligo-, bi- and unipotent progenitors. However, this model is based on the analysis of predefined flow-sorted cell populations. Here we integrated flow cytometric, transcriptomic and functional data at single-cell resolution to quantitatively map early differentiation of human HSCs towards lineage commitment. During homeostasis, individual HSCs gradually acquire lineage biases along multiple directions without passing through discrete hierarchically organized progenitor populations. Instead, unilineage-restricted cells emerge directly from a ‘continuum of low-primed undifferentiated haematopoietic stem and progenitor cells’ (CLOUD-HSPCs). Distinct gene expression modules operate in a combinatorial manner to control stemness, early lineage priming and the subsequent progression into all major branches of haematopoiesis. These data reveal a continuous landscape of human steady-state haematopoiesis downstream of HSCs and provide a basis for the understanding of haematopoietic malignancies.

Clonal evolution is believed to be a main driver for progression of various types of cancer and implicated in facilitating resistance to drugs. However, the hierarchical organization of malignant clones in the hematopoiesis of myelodysplastic syndromes (MDS) and its impact on response to drug therapy remain poorly understood. Using high-throughput sequencing of patient and xenografted cells, we evaluated the intratumoral heterogeneity (n= 54) and reconstructed mutational trajectories (n = 39) in patients suffering from MDS (n = 52) and chronic myelomonocytic leukemia-1 (n = 2). We identified linear and also branching evolution paths and confirmed on a patient-specific level that somatic mutations in epigenetic regulators and RNA splicing genes frequently constitute isolated disease-initiating events. Using high-throughput exome- and/or deep-sequencing, we analyzed 103 chronologically acquired samples from 22 patients covering a cumulative observation time of 75 years MDS disease progression. Our data revealed highly dynamic shaping of complex oligoclonal architectures, specifically upon treatment with lenalidomide and other drugs. Despite initial clinical response to treatment, patients’ marrow persistently remained clonal with rapid outgrowth of founder-, sub-, or even fully independent clones, indicating an increased dynamic rate of clonal turnover. The emergence and disappearance of specific clones frequently correlated with changes of clinical parameters, highlighting their distinct and far-reaching functional properties. Intriguingly, increasingly complex mutational trajectories are frequently accompanied by clinical progression during the course of disease. These data substantiate a need for regular broad molecular monitoring to guide clinical treatment decisions in MDS.

Clonal evolution is believed to be a main driver for progression of various types of cancer and implicated in facilitating resistance to drugs. However, the hierarchical organization of malignant clones in the hematopoiesis of myelodysplastic syndromes (MDS) and its impact on response to drug therapy remain poorly understood. Using high-throughput sequencing of patient and xenografted cells, we evaluated the intratumoral heterogeneity (n= 54) and reconstructed mutational trajectories (n = 39) in patients suffering from MDS (n = 52) and chronic myelomonocytic leukemia-1 (n = 2). We identified linear and also branching evolution paths and confirmed on a patient-specific level that somatic mutations in epigenetic regulators and RNA splicing genes frequently constitute isolated disease-initiating events. Using high-throughput exome- and/or deep-sequencing, we analyzed 103 chronologically acquired samples from 22 patients covering a cumulative observation time of 75 years MDS disease progression. Our data revealed highly dynamic shaping of complex oligoclonal architectures, specifically upon treatment with lenalidomide and other drugs. Despite initial clinical response to treatment, patients' marrow persistently remained clonal with rapid outgrowth of founder-, sub-, or even fully independent clones, indicating an increased dynamic rate of clonal turnover. The emergence and disappearance of specific clones frequently correlated with changes of clinical parameters, highlighting their distinct and far-reaching functional properties. Intriguingly, increasingly complex mutational trajectories are frequently accompanied by clinical progression during the course of disease. These data substantiate a need for regular broad molecular monitoring to guide clinical treatment decisions in MDS.

Hematopoietic stem and progenitor cells (HSPCs) are responsible for the production of blood and immune cells. Throughout life, HSPCs acquire oncogenic aberrations that can cause hematological cancers. Although molecular programs maintaining stem cell integrity have been identified, safety mechanisms eliminating malignant HSPCs from the stem cell pool remain poorly characterized. Here, we show that HSPCs constitutively present antigens via major histocompatibility complex class II. The presentation of immunogenic antigens, as occurring during malignant transformation, triggers bidirectional interactions between HSPCs and antigen-specific CD4+T cells, causing stem cell proliferation, differentiation, and specific exhaustion of aberrant HSPCs. This immunosurveillance mechanism effectively eliminates transformed HSPCs from the hematopoietic system, thereby preventing leukemia onset. Together, our data reveal a bidirectional interaction between HSPCs and CD4+T cells, demonstrating that HSPCs are not only passive receivers of immunological signals but also actively engage in adaptive immune responses to safeguard the integrity of the stem cell pool.

Around birth, hematopoietic stem cells (HSCs) expanding in the fetal liver migrate to the developing bone marrow (BM) to mature and expand. To identify the molecular processes associated with HSCs located in the 2 different microenvironments, we compared the expression profiles of HSCs present in the liver and BM of perinatal mice. This revealed the higher expression of a cluster of extracellular matrix-related genes in BM HSCs, with secreted protein acidic and rich in cysteine (SPARC) being one of the most significant ones. This extracellular matrix protein has been described to be involved in tissue development, repair, and remodeling, as well as metastasis formation. Here we demonstrate that SPARC-deficient mice display higher resistance to serial treatment with the chemotherapeutic agent 5-fluorouracil (5-FU). Using straight and reverse chimeras, we further show that this protective effect is not due to a role of SPARC in HSCs, but rather is due to its function in the BM niche. Although the kinetics of recovery of the hematopoietic system is normal, HSCs in a SPARC-deficient niche show an accelerated return to quiescence, protecting them from the lethal effects of serial 5-FU treatment. This may become clinically relevant, as SPARC inhibition and its protective effect on HSCs could be used to optimize chemotherapy schemes.

Around birth, hematopoietic stem cells (HSCs) expanding in the fetal liver migrate to the developing bone marrow (BM) to mature and expand. To identify the molecular processes associated with HSCs located in the 2 different microenvironments, we compared the expression profiles of HSCs present in the liver and BM of perinatal mice. This revealed the higher expression of a cluster of extracellular matrix-related genes in BM HSCs, with secreted protein acidic and rich in cysteine (SPARC) being one of the most significant ones. This extracellular matrix protein has been described to be involved in tissue development, repair, and remodeling, as well as metastasis formation. Here we demonstrate that SPARC-deficient mice display higher resistance to serial treatment with the chemotherapeutic agent 5-fluorouracil (5-FU). Using straight and reverse chimeras, we further show that this protective effect is not due to a role of SPARC in HSCs, but rather is due to its function in the BM niche. Although the kinetics of recovery of the hematopoietic system is normal, HSCs in a SPARC-deficient niche show an accelerated return to quiescence, protecting them from the lethal effects of serial 5-FU treatment. This may become clinically relevant, as SPARC inhibition and its protective effect on HSCs could be used to optimize chemotherapy schemes.

Previous studies have established pivotal roles for c-Myc and its homolog N-Myc in hematopoietic stem cell (HSC) maintenance and niche-dependent differentiation. However, it remains largely unclear how c-Myc expression is regulated in this context. Here, we show that HSCs and more committed progenitors express similar levels of c-myc transcripts. Using knock-in mice expressing a functional enhanced green fluorescent protein-c-Myc fusion protein under control of the endogenous c-myc locus, c-Myc protein levels were assessed. Although HSCs express low levels of c-Myc protein, its expression increases steadily during progenitor differentiation. Thus, mRNA and protein expression patterns differ significantly in stem/progenitor cells, suggesting that c-Myc expression is largely controlled posttranscriptionally. Moreover, interferon-α exposure, which activates dormant HSCs, strongly induces c-Myc expression at the protein level but not at the transcript level. This posttranscriptional mechanism of c-Myc regulation provides the blood system with a rapid way to adjust c-Myc expression according to demand during hematopoietic stress.

Previous studies have established pivotal roles for c-Myc and its homolog N-Myc in hematopoietic stem cell (HSC) maintenance and niche-dependent differentiation. However, it remains largely unclear how c-Myc expression is regulated in this context. Here, we show that HSCs and more committed progenitors express similar levels of c-myc transcripts. Using knock-in mice expressing a functional enhanced green fluorescent protein-c-Myc fusion protein under control of the endogenous c-myclocus, c-Myc protein levels were assessed. Although HSCs express low levels of c-Myc protein, its expression increases steadily during progenitor differentiation. Thus, mRNA and protein expression patterns differ significantly in stem/progenitor cells, suggesting that c-Myc expression is largely controlled posttranscriptionally. Moreover, interferon-α exposure, which activates dormant HSCs, strongly induces c-Myc expression at the protein level but not at the transcript level. This posttranscriptional mechanism of c-Myc regulation provides the blood system with a rapid way to adjust c-Myc expression according to demand during hematopoietic stress.

Durie: Amgen, Celgene, Johnson & Johnson, and Takeda: Consultancy. Raab:Novartis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Heidelberg Pharma: Research Funding. Müller-Tidow:Pfizer: Research Funding, Speakers Bureau; Daiichi Sankyo: Research Funding; BiolineRx: Research Funding; Janssen-Cilag GmbH: Speakers Bureau. Goldschmidt:BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Research Funding; Merck Sharp and Dohme (MSD): Research Funding; Johns Hopkins University: Other: Grants and/or provision of Investigational Medicinal Product; Dietmar-Hopp-Foundation: Other: Grants and/or provision of Investigational Medicinal Product:; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product, Research Funding; Incyte: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline (GSK): Honoraria; University Hospital Heidelberg, Internal Medicine V and National Center for Tumor Diseases (NCT), Heidelberg, Germany: Current Employment; Chugai: Honoraria, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Molecular Partners: Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; Mundipharma GmbH: Research Funding.

To date, multiple myeloma (MM) remains an incurable disease with only a minor fraction of patients experiencing long-term remission (LTR) over 7 years after a single therapy line. Myeloma cells strongly depend on the interaction with their bone marrow microenvironment (BMME), but the molecular and cellular adaptations of the BMME to active MM disease and the role of the immune system in patients experiencing LTR remain poorly understood. In order to gain a global and detailed understanding of the BMME, we profiled over 290.000 BM resident cells from 11 MM patients in LTR 7 to 17 years after first-line therapy and 3 healthy donors using droplet-based single-cell RNA sequencing. Paired BM samples collected at initial diagnosis enabled us to analyze the changes from first diagnosis to the state of LTR in individual patients.

Invasive pulmonary aspergillosis (IPA) is a life-threatening infectious complication mainly affecting immunocompromised hematological patients. Therapeutic success requires early and precise diagnostics. Few data are available on the performance of current diagnostic tests in patients admitted to the intensive care unit (ICU) as the majority of diagnostic studies is conducted in hematological patients and results cannot readily be transferred to patients in the ICU setting.

No relevant conflicts of interest to declare.

Fricke: Apogenix AG: Employment. Kunz:Apogenix AG: Employment.

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Introduction:Myelodysplastic Syndrome (MDS) can occur in young people but it is mainly a disease of the elderly with a dramatic increase of incidence in the decades above 60 years. Accordingly, the factor age may be an important gateway to the understanding of the molecular pathogenesis of MDS. Insights into the molecular changes of aging hematopoiesis in healthy organisms have found molecular changes, which often parallel the observations in MDS such as increase of clonality with age, change of epigenetic profiles, skewed lineage commitment toward the myeloid compartment and reduced regenerative capacity after stress. The development of MDS is often suggestive of an accelerated extrapolation of molecular changes, which also occur in normal aging hematopoiesis. Beyond this, increasing evidence is suggesting that MDS hematopoiesis is highly dependent on support of the bone marrow (BM) stroma, which has been shown to display aberrant transcriptomic profiles as compared to healthy BM stroma. To this end, we aimed to test the hypothesis whether the emergence of MDS may be associated with a continuity of molecular changes in BM stroma cells during aging. Therefore, we performed explorative RNA sequencing in a set of MSCs collected from healthy young, healthy old and patients with MDS with a highly standardized pre-analytical work-up algorithm.

No relevant conflicts of interest to declare.

Bertz: GILEAD Sciences: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Buchheidt:Astellas: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; MSD: Consultancy, Honoraria; Gilead: Consultancy, Honoraria.

Buchheidt: Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Other: travel grant, Research Funding, Speakers Bureau; Astellas: Other: travel grant, Speakers Bureau; Merck Sharp & Dohme: Other: travel grant, Speakers Bureau.

Introduction

Introduction

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